1. Field of the Invention
The present invention relates to lateral flow test strips and methods of operation for the lateral flow test strips.
2. Description of Related Art
Quantitative analysis of cells and analytes in fluid samples, particularly bodily fluid samples, often provides critical diagnostic and treatment information for physicians and patients. For example, immunological testing methods which take advantage of the high specificity of antigen-antibody reactions, provide one approach to measurement of analytes. Kennedy, D. M. and S. J. Challacombe, eds., ELISA and Other Solid Phase Immunoassays: Theoretical and Practical Aspects, John Wiley and Sons, Chichester (1988). This document and all others cited to herein, are incorporated by reference as if reproduced fully below. Such assays may also find use in various other applications, such as veterinary, food testing, or agricultural applications.
Immunoassays that provide a quantitative measurement of the amount of an analyte in a sample have previously used complex, multi-step procedures and expensive analyzers available only in a laboratory setting.
Immunochromatographic assays, such as those described in GB 2,204,398A; U.S. Pat. Nos. 5,096,837, 5,238,652, and 5,266,497; Birnbaum, S. et al., Analytical Biochem. 206:168-171 (1992); Roberts, M. A. and R. A. Durst, Analytical Chem. 67:482-491 (1995); and Klimov, A. D. et al., Clinical Chem. 41:1360 (1995), are simpler, yet do not provide a quantitative measurement of an analyte. Instead, these immunochromatographic assays detect the presence (or absence) of an analyte above a defined cutoff level for the test performed. The lack of a quantitative measurement limits the usefulness of these assays.
A variety of disposable diagnostic assay devices have also been developed. Examples of such devices include, but are not limited to Cathey, et al, U.S. Pat. No. 5,660,993; International Publication Number WO 92/12428; Eisinger, et al, U.S. Pat. No. 4,943,522; Campbell, et al, U.S. Pat. No. 4,703,017; Campbell, et al, U.S. Pat. No. 4,743,560; and Brooks, U.S. Pat. No. 5,753,517. Nevertheless, a need still exists for improved disposable diagnostic assay devices and methods.
Test strips are provided which are adapted to receive a buffer that prewets the test strip and receive a sample which flows within the prewet test strip. The test strips are employed to detect one or more analytes that may be present in a sample.
According to one embodiment, the test strip comprises a buffer addition zone to which a buffer is added to prewet the test strip; an absorbent zone proximal to the buffer addition zone; one or more test zones distal to the buffer addition zone, at least one of the test zones including a first analyte binding agent immobilized therein which is capable of binding to the analyte to be detected; and a terminal buffer flow zone distal to the one or more test zones, the absorbent zone being positioned relative to the buffer addition zone and having an absorption capacity relative to the other zones of the test strip such that when a volume of buffer within a predetermined buffer volume range for the test strip is added to the buffer addition zone, a distal diffusion front of the buffer diffuses from the buffer addition zone to a distal diffusion point within the terminal buffer flow zone and then diffuses proximal relative to the one or more test zones. The test strip further comprises a sample addition zone that is distal to the terminal buffer flow zone. When a sample is added to the sample addition zone, the sample diffuses within the test strip in a proximal direction across the terminal buffer flow zone, across the one or more test zones, and ultimately to the absorbent zone. When the sample traverses the test zones, analyte in the sample is immobilized in whichever test zone(s) include(s) the first analyte binding agent bound therein.
The above described test strip may be used to detect an analyte in a sample by a direct detection assay or may be used to detect an analyte in a sample by a competitive assay. When the assay is a direct detection assay, the amount of analyte in the sample is measured based on the amount of analyte which is immobilized in a test zone by a first analyte binding agent bound therein. When the assay is a competitive assay, the test strip further comprises a competitive agent which is capable of competing with the analyte for binding to the first analyte binding agent. In this instance, the amount of analyte in the sample is measured based on how much less competitive agent is immobilized in the test zone by the first analyte binding agent as compared to when a control is employed as the sample which contains no analyte.
Control over the above described flow of the buffer within the test strip (i.e., such that the buffer reaches the terminal buffer flow zone and reverses the direction of buffer flow within the terminal buffer flow zone back toward the buffer addition zone and the absorbent zone) is achieved by controlling the amount of buffer added to the test strip within a predetermined range designed to be used with that test strip.
By adding the sample to the sample addition zone such that the sample reaches the terminal buffer flow zone after the buffer has reached the terminal buffer flow zone and has already reversed direction and is diffusing back toward the absorbent zone, the sample is able to flow within a prewet test strip, thereby yielding more accurate and precise results.
As will be described in greater detail herein, depending on the layout of the test strip, the buffer may be added before, at the same time, or after the sample is added to the test strip. For example, the sample addition zone may be positioned relative to the test zones such that sample is added to the sample addition zone at the same time that buffer is added to the buffer addition zone. The sample addition zone may also be positioned relative to the test zones such that sample added to the sample addition zone at the same time that the buffer is added to the buffer addition zone. The sample addition zone may also be positioned relative to the test zones such that the sample can be added to the test strip before the buffer is added and nevertheless, the sample still reaches the distal diffusion point of the buffer after the distal diffusion front of the buffer has diffused to the distal diffusion zone, reversed direction and begun diffusing in a proximal direction.
According to any of the above test strip embodiments, 1, 2, 3 or more test zones may be control zones with one or more control binding agents immobilized therein. The control zones may be used to calibrate the test strip, may be used to confirm whether or not the test strip performed as intended, may be used detect whether too little or too much buffer was added and may be used to detect whether too little sample was added.
In one embodiment, the test strip comprises at least a first control zone with a control binding agent immobilized therein. Optionally, the test zones further includes a second control zone with a same control binding agent immobilized therein as the first control zone. The first control zone may contain the same or a different amount of the control binding agent than the second control zone. In a preferred embodiment, the first control zone contains about the same amount of the control binding agent as the second control zone.
Also according to any of the above test strip embodiments, a second analyte binding agent which is capable of binding to the analyte and diffusing to the one or more test zones may be included on the test strip. The second analyte binding agent is preferably incorporated on the test strip adjacent either the sample addition zone or the buffer addition zone, more preferably proximal relative to the sample addition zone or distal relative to the buffer addition zone such that addition of the sample or buffer causes the second analyte binding agent to be carried with the sample or buffer to the test zones.
The second analyte binding agent may also be delivered to the test strip via the buffer or the sample, most preferably the sample. The second analyte binding agent may bind to components in the sample in addition to the analyte. Alternatively, the second analyte binding agent may be an agent which does not bind to components in the sample other than the analyte.
In order to facilitate detection, the second analyte binding agent is preferably labeled with a detectable marker. As discussed herein, any of a wide range of detectable markers known in the art may be used. In a preferred embodiment, the second analyte binding agent is attached to a particle which is capable of diffusing to the one or more test zones. The particle may serve as the detectable marker or may itself be labeled with a detectable marker.
Also according to any of the above test strip embodiments, the test strip may be for a competitive assay, in which case, the test strip may include a competitive agent. The competitive agent may compete with the analyte for binding to the first analyte binding agent.
The competitive agent is preferably incorporated on the test strip adjacent the sample addition zone, more preferably proximal relative to the sample addition zone such that addition of the sample causes the competitive agent to be carried with the sample to the test zone.
Methods are also provided for detecting an analyte in a sample.
In one embodiment, the method comprises delivering a buffer to a test strip which causes a distal diffusion front of the buffer to (a) diffuse in a distal direction to one or more test zones, at least one of the test zones including a first analyte binding agent immobilized therein which binds to analyte in the sample, (b) diffuse to a terminal buffer flow zone distal to the one or more test zones, change direction and (c) diffuse to a position proximal to the one or more test zones; delivering a sample to the test strip at a position distal to the terminal buffer flow zone, delivery of the sample causing analyte in the sample to diffuse proximally past the terminal buffer flow zone to the one or more test zones after the distal diffusion front of the buffer diffuses proximal to the one or more test zones, the analyte binding to the first analyte binding agent and becoming immobilized in the test zones; and detecting the analyte immobilized in the test zones.
According to the method, a second analyte binding agent may be present which binds to the analyte. The second analyte binding agent may be used to detect the immobilized analyte. The second analyte binding agent may be contained on the test strip where the sample is delivered, delivery of the sample causing the diffusion of the second analyte binding agent. Alternatively, the second analyte binding agent may be contained on the test strip proximal to where the sample is delivered, delivery of the sample causing the diffusion of the second analyte binding agent. Delivering the sample to the test strip may also include delivering the second analyte binding agent to the test strip within the sample.
In another embodiment, the method is for a competitive assay. According to this method, a buffer is delivered to a test strip which causes a distal diffusion front of the buffer to (a) diffuse in a distal direction to one or more test zones, at least one of the test zones including a first analyte binding agent immobilized therein which binds to analyte in the sample, (b) diffuse to a terminal buffer flow zone distal to the one or more test zones, change direction and (c) diffuse to a position proximal to the one or more test zones. A sample is also delivered to the test strip at a position distal to the terminal buffer flow zone such that delivery of the sample causes the sample diffuse proximally past the terminal buffer flow zone to the one or more test zones after the distal diffusion front of the buffer diffuses proximal to the one or more test zones.
Delivery of a sample to the test strip also causes a competitive agent to diffuse with the sample to the test zone. The competitive agent competes with the analyte for binding to the first analyte binding agent. The competitive agent is preferably incorporated on the test strip adjacent the sample addition zone, more preferably proximal relative to the sample addition zone such that addition of the sample causes the competitive agent to be carried with the sample to the test zone.
The method further comprises detecting the competitive agent immobilized in the test zones. In order to facilitate detection, the competitive agent is preferably labeled with a detectable marker.
According to any of the method embodiments, the buffer may be added to the test strip at a same time as the sample is added to the test strip, before the sample is added to the test strip, or after the sample is added to the test strip. When the sample is added to the test strip relative to the conjugate buffer depends on the time required for the buffer to reach the terminal buffer flow zone which, in turn, depends on the flow design of the test strip.
According to any of the above methods, the test zones may include a first control zone with a control binding agent immobilized therein, delivering the buffer causing a control agent to diffuse distally to the first control zone and bind to the control binding agent immobilized therein. Alternatively, the test zones may include first and second control zones which each include an approximately the same or significantly different amount of a control binding agent immobilized therein, delivering the buffer causing a control agent to diffuse distally to the first and second control zones and bind to the control binding agent immobilized therein.
When one or more control zones are employed, a control agent may be contained on the test strip where the buffer is delivered, delivery of the buffer causing the diffusion of the control agent. Alternatively, a control agent may be contained on the test strip distal to where the buffer is delivered, delivery of the buffer causing the diffusion of the control agent. Delivering the buffer to the test strip may also include delivering the control agent to the test strip within the buffer. Incorporating the control agent into the buffer is advantageous because variability in the movement of control agents strip to strip arising from differences in the way in which the control agents becomes resolubilized when buffer is added is avoided.
Also according to the above methods, detecting the second analyte binding agent may be facilitated by labeling the second analyte binding agent with a detectable marker, detecting the second analyte binding agent including detecting the detectable marker. The second analyte binding agent may be attached to a particle. Detecting the second analyte binding agent may include detecting the particle.
According to any of the above embodiments, the buffer delivered to the test strip is preferably within a predetermined volume range that the test strip has been designed to process. The predetermined volume range is preferably between about 10 and 250 xcexcL, preferably between about 20 and 200 xcexcL, more preferably between about 20 and 100 xcexcL, and most preferably between about 40 and 60 xcexcL. When a buffer is delivered to the test strip within the predetermined volume range, the terminal sample flow zone may be designed to have a short length from a proximal end to a distal end. For example, when a buffer is delivered to the test strip within a range of about 35 and 45 xcexcL, the terminal flow zone may have a length from a proximal end to a distal end of between about 1 and 25 mm, more preferably 2 and 15 mm, and most preferably 3 and 10 mm.
Also according to any of the above embodiments, the first analyte binding agent preferably does not bind to components in the sample other than the analyte. Types of molecules that can serve as first analyte binding agents include, but are not limited to antibodies, engineered proteins, peptides, haptens, lysates containing heterogeneous mixtures of antigens having analyte binding sites, ligands and receptors. In one particular embodiment, the first analyte binding agent is an antibody or fragment thereof.
Also according to any of the above embodiments, the buffer added to the buffer addition zone may comprise the sample being tested. Optionally, the buffer may be the sample. When sample forms all or a portion of the buffer that is added to buffer addition zone, the buffer still performs the function of prewetting the test strip. The ability to use sample, in whole or in part, as the buffer allows the present invention to more easily accommodate a wider range of sample and external liquid control matrices (e.g., serum, plasma, euglobulin). In addition, differences in flow behavior within the test strip between sample and buffer can be reduced by adding the same composition (e.g., the sample) to both the sample and buffer addition zones.